Dissertations / Theses on the topic 'Megakaryocytes'
To see the other types of publications on this topic, follow the link: Megakaryocytes.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Megakaryocytes.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Melford, Steven K. "Calcium signalling in megakaryocytes." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364104.
Full text
2
Hamlet, Isla. "GATA1 protein partners in megakaryocytes." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442463.
Full text
3
McGrath, Catherine Jane. "Glutamate release mechanisms from megakaryocytes." Thesis, University of York, 2007. http://etheses.whiterose.ac.uk/9950/.
Full textAbstract:
Cardiovascular disease (CVD) is one of the main causes of death in western society. Platelet activation, thrombus formation and plaque rupture are all central events in the pathogenesis of acute coronary syndromes, therefore therapies targeted at controlling platelet numbers and aggregation are likely to be beneficial in the treatment of CVD. Megakaryocytes (MKs) which are the precursors to platelets are an ideal target for these therapies, however the intrinsic factors that regulate the production and shedding of platelet precursors are poorly understood. Recent studies identified that MKs express functional NMDA-type glutamate receptors similar to those found in the CNS and that antagonism of these receptors prevents normal MK differentiation and platelet function. This thesis investigates glutamate signalling within MKs further, focusing on the glutamate release capability of MK cells and the mechanisms involved. Using molecular and cellular techniques it was demonstrated that MK cells expressed numerous regulatory proteins required for vesicular glutamate release, including core SNARE proteins, VAMP, SNAP-23 and syntaxin; specific glutamate-loading vesicle proteins, VGLUTI and VGLUT2; and glutamate transporters, EAATI and EAAT2. Active vesicle recycling was observed in MK cells using a fluorescent reporter and an enzyme-linked fluorimetric assay confirmed that MK cells constitutively released glutamate and that glutamate release levels increased significantly following MK differentiation. Transient transfection of the human cell line MEG-Ol with tetanus toxin, which disables vesicle recycling, induced a 30% decrease (P
4
Cowart, Miles. "Exploring the role of NIPSNAP4, CIB1, and PLK3 in megakaryocyte maturation." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 80 p, 2007. http://proquest.umi.com/pqdweb?did=1253509361&sid=2&Fmt=2&clientId=8331&RQT=309&VName=PQD.
Full text
5
Kuhl, Christiane. "Structure / function analysis of GATA1 in megakaryocytes." Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.418523.
Full text
6
Sobolewski, S. "A new function of megakaryocytes in malignancies." Thesis, University of Bradford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.375083.
Full text
7
Hitchcock, Ian Stuart. "Functional activity of NMDA receptors on megakaryocytes." Thesis, University of York, 2003. http://etheses.whiterose.ac.uk/9856/.
Full text
8
Dalby, Amanda Louise. "Forward programming of human pluripotent stem cells to a megakaryocyte-erythrocyte bi-potent progenitor population : an in vitro system for the production of platelets and red blood cells for transfusion medicine." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/273749.
Full textAbstract:
There exists a need to produce platelets in vitro for use in transfusion medicine, due to increased platelet demands and short shelf life. Our lab uses human induced pluripotent stem cells (iPSCs), as an attractive alternative supply, as iPSCs can be cultured indefinitely and differentiate into almost any cell type. Using a technique called forward programming, we over express three key haematological transcription factors (TFs), pushing iPSCs towards the megakaryocyte lineage, to produce mature megakaryocytes, the platelet precursor cell type. A major limitation of the forward programming technique is a reliance of lentiviral transduction to overexpress the three TFs, which leads to a number of issues including heterogeneity and high experimental costs. To overcome this, I have developed an inducible iPSC line by inserting the forward programming TFs into a genomic safe harbour, using genome editing techniques. TF expression is strictly controlled, with the TFs expressed only after chemical induction. Inducing forward programming is an efficient method for producing mature megakaryocytes and these cells maintain higher purity in long-term cultures, when compared to cells produced by the lentiviral method. Removing the requirement of lentiviral transduction is a major advancement, making forward programming more amenable to scaling-up, thus moving this technology closer towards our goal of producing in vitro platelets for use in transfusion medicine. I have also shown that forward programming generates a bi-potent progenitor population, from which erythroblasts can be generated, by altering only media conditions. As for megakaryocyte cultures, inducing forward programming improves the purity of erythroblasts produced, compared to the lentiviral method. I have developed single cell progenitor assays combined with index sorting of different cell surface markers, to allow retrospective analysis of cells which successfully generate colonies. The aim of this work is to better characterise the progenitor cells produced by forward programming, to allow further study of this cell type. Single cell RNA-seq of megakaryocytes revealed heterogeneity in long-term cultures and also identified novel candidate surface markers that may help to further characterise the progenitor cell population.
9
Wagner, Leonard [Verfasser], and Andreas [Gutachter] Geier. "Zinc homeostasis in megakaryocytes / Leonard Wagner ; Gutachter: Andreas Geier." Würzburg : Universität Würzburg, 2020. http://d-nb.info/1220634220/34.
Full text
10
Abdalla, Sarah. "Identification of the Regions in Factor V Mediating its Edocytosis by Megakaryocytes to Form the Unique Platelet-Derived Cofactor Molecule." ScholarWorks @ UVM, 2013. http://scholarworks.uvm.edu/graddis/6.
Full textAbstract:
Factor Va is a plasma protein that plays an important role in the regulation of blood coagulation by serving as the essential cofactor in thrombin generation via the prothrombinase complex. The procofactor, factor V, exists in two whole blood pools with 75-80% found in plasma, and 20-25% stored in the α-granules of platelets. As compared to the plasma procofactor, platelet-derived factor V is physically and functionally distinct, and displays a more procoagulant phenotype. Despite these profound differences, platelet-derived factor V originates via endocytosis of the plasma-derived procofactor by megakaryocytes. Endocytosis is mediated by two receptors: an unidentified, specific factor V receptor, and low density lipoprotein (LDL) receptor related protein-1 (LRP-1), a ubiquitous receptor that plays a role in endocytosis of proteins targeted for lysosomal degradation. These observations represent a novel role for LRP-1 in endocytosis of a protein that is functionally modified, and not targeted for lysosomal degradation. The goal of this study is to define the factor V regions involved in its interactions with the unidentified factor V receptor and LRP-1 expressed on megakaryocytes to begin to elucidate the molecular mechanisms regulating formation of the unique platelet-derived cofactor. Epitope mapping studies were performed using anti-factor V monoclonal antibodies, E9 and anti-factor V #2. Previous observations indicated that these factor Va light chain antibodies inhibited endocytosis of factor V by megakaryocytes. However, subsequent analyses demonstrated that only E9 inhibited both factor V binding and endocytosis. Thus, it was used for these studies. Western blotting of factor V and Va suggested that E9 recognizes a conformation-dependent epitope, which precluded the use of conventional epitope mapping approaches used for linear epitopes. E9 had no effect on factor Va cofactor activity in a plasma-based clotting assay suggesting that it does not perturb factor Va’s interactions with the membrane surface or factor Xa. Cleavage of lipid-bound factor Va by factor Xa at Arg1765 was also not affected by the presence of E9 suggesting that the epitope is not directed against this cleavage site. When E9 was used to immunoprecipitate the factor Xa-generated light chain cleavage products, both the 48/46 and 30 kDa light chain fragments were captured. These observations were confirmed using a solid phase competition assay where factor Xa-cleaved factor Va inhibited binding of 125I-factor V to E9 as well as intact factor V or Va. Limited proteolysis of the factor Va light chain with trypsin or Asp-N, generated products that were no longer detectable in this assay. These combined observations suggest that the anti-factor V light chain antibody, E9, has an epitope that is conformation-dependent and extremely labile. Future directions and alternative approaches are discussed.
11
Shah, Chirag Manubhai. "Mechanisms of nitric oxide transfer from s-nitrosothiols into megakaryocytes." Thesis, University of Westminster, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433625.
Full text
12
Cheung, Manyee. "Investigation of megakaryocytes from normal and myeloproliferative bone marrow biopsies." Thesis, University of Southampton, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343012.
Full text
13
Frydman, Galit Hocsman. "The role of megakaryocytes and platelets in infection and immunothrombosis." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/115756.
Full textAbstract:
Thesis: Sc. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018.Cataloged from PDF version of thesis. Includes CD-ROM with 9 videos in the .avi format and 2 video in the .mp4 format.
Includes bibliographical references.
Megakaryocytes (MKs), one of the largest and rarest hematopoietic stem cells in the bone marrow, have traditionally played a primary role in hemostasis as precursors to platelets, which are importantly, one of the most abundant cell types in the peripheral circulation. While platelets are studied for their various roles in inflammation, the role of MKs within the innate immune system has not been explored. In a series of comprehensive in vitro experiments, we have demonstrated that both cord blood-derived MKs and MKs from a megakaryoblastic lineage have innate immune cell functions, including: phagocytosis, formation of extracellular traps, and chemotaxis towards pathogenic stimuli. MKs were also observed to directionally release platelets towards pathogenic stimuli. In addition to their primary role as immune cells, MKs were also shown to contain extranuclear histones, which the MKs release along with budding platelets into the circulation. These small packages of histones can play a major role in inflammation and immunothrombosis by promoting inflammation and coagulation. By evaluating blood and tissue samples from patients diagnosed with sepsis, we demonstrated that there is an increased MK concentration both in the peripheral circulation, as well as in the lungs and kidneys. Platelets from patients with sepsis also appeared to have a specific phenotype, including increased DNA and histone staining. MK number in the circulation and end-organs, as well as platelet histone expression appeared to be correlated with both prognosis and type of infection. This newly recognized role of MKs as functional innate immune cells may have significant implications for the role of MKs in conditions such as sepsis and, pending a more profound mechanistic understanding, may further lead to the development of novel targets for the treatment of sepsis.
by Galit Hocsman Frydman.
Sc. D.
14
Линдіна, Юлія Миколаївна, Юлия Николаевна Лындина, Yuliia Mykolaivna Lyndina, Владислав Володимирович Сікора, Владислав Владимирович Сикора, Vladyslav Volodymyrovych Sikora, Наталія Іванівна Гирявенко, et al. "Peculiarities of megakaryocytes and platelets variation in the microelementosis condition." Thesis, Springer, 2019. http://essuir.sumdu.edu.ua/handle/123456789/75110.
Full textAbstract:
Background & Objectives: The aim of our study was to characterise megakaryocytes and platelets variation under the influence of heavy metal salts and determine the peculiarities of these changes in recovery period. Methods: The laboratory rats (n=48) were used for the conduction of experimental investigation. They were divided into two groups: control – animals which drank clean water and experimental – rats which drank water with heavy metal salts (Zn, Cu, Fe, Mn, Cr and Pb). The bone marrow structure and blood parameters were studied on 30, 90, 120 and 180 day. Results: During 90 days of intoxication the amount of megakaryocytes increased on 40%(р=0.017). There were changes of shape, size (small and giant), nuclear-cytoplasmic ratio, polysegmentation of nuclei and loss of megakaryocyte-sinusoid contacts. It was found the platelets increase by 11% in the blood. Suspending the addition of pollutants to the animal's diet was accompanied by progressive restoration of bone marrow-blood parameters. Simultaneously with disappearance of megakaryocytes morphological variations the area of thrombocytopoiesis was decreased by 28%, the thrombocytes – by 5.3%. Conclusion: The intake of heavy metal salts in elevated concentrations provokes significant disorders in thrombocytopoiesis which leads for platelets increase in the blood. Although there was a significant improvement bone marrow-blood characteristics during recovery period, their values do not reach the indicators of the intact group animals.
15
Taylor, Kirk Allen. "Pannexin-1 and other anion channels in platelets and megakaryocytes." Thesis, University of Leicester, 2015. http://hdl.handle.net/2381/31997.
Full textAbstract:
Platelet ion channels are essential for Ca2+-influx, maintaining the resting membrane potential and cell volume regulation. Cation channels have been widely studied but few reports of platelet anion channels exist. A recent ‘channelome’ screen has suggested that human platelets express several anion-permeable channels of unknown function. This thesis explores the function of two such channels, Pannexin-1 and TMEM16F, in platelets, primary megakaryocytes (MKs) and related cell lines. Using pannexin-1 inhibitors, these channels were shown to open in response to stimulation by thrombin, contribute to Ca2+-influx and release cytosolic ATP following stimulation by threshold concentrations of platelet agonists. Experiments also suggested that ATP release by pannexin-1 channels contributes to Ca2+-influx via stimulation of ATP-gated P2X1 receptors. Anion-selective TMEM16F channels have been recorded in a variety of cell types and activate in response to sustained elevation of [Ca2+]i to 100 μM. Controversially, these channels were reported to be cation-selective in mouse MKs. Thus, whole cell patch clamp recordings were performed to assess the biophysical properties of TMEM16F channels in HEL cells and primary mouse and rat MKs. Elevating [Ca2+]i to 100 μM in HEL cells and rat MKs induced a Ca2+-dependent, outwardly rectifying anion-permeable conductance, which was blocked by the TMEM16F inhibitor A01. Recordings of mouse MKs identified an equally Ca2+-dependent, outwardly rectifying and A01-sensitive conductance, however this was predominantly permeable to cations. Thus, a major interspecies difference exists in the ionic selectivity of MK TMEM16F channels; possible explanations for this difference, such as mutations within the pore region, are discussed. In summary, this thesis has explored the biophysical properties and function of platelet and MK anion channels using in vitro assays. These studies have relied heavily upon pharmacological tools and future studies of platelet function would benefit from the use of transgenic models.
16
Gertz, Jacqueline Michelle. "Cellular And Molecular Events Regulating Factor V Endocytosis By Megakaryocytes." ScholarWorks @ UVM, 2015. http://scholarworks.uvm.edu/graddis/401.
Full textAbstract:
Platelet- and plasma-derived factor Va are absolutely essential for thrombin generation catalyzed by the prothrombinase complex, a 1:1 stoichiometric complex of the serine protease factor Xa and the nonenzymatic cofactor, factor Va, assembled on an appropriate membrane surface in the presence of calcium ions. Two whole blood pools of the procofactor, factor V, exist: approximately 75% circulates in the plasma as a single chain inactive molecule, while the other 25% resides in platelet α-granules in a partially proteolytically-activated state. Our laboratory demonstrated that the platelet-derived cofactor originates following endocytosis of plasma-derived factor V by megakaryocytes, the platelet precursor cells, via a two receptor system including an uncharacterized, specific factor V receptor and low density lipoprotein receptor related protein-1. Following endocytosis factor V is physically and functionally modified and trafficked to the platelet α-granule from where it is released upon platelet activation at sites of vascular injury.
The first goal of this dissertation was to define how factor V endocytosis changes over the course of megakaryocyte development. Hematopoietic multipotential stem cells were isolated from human umbilical cord blood and subjected to ex vivo differentiation into megakaryocytes. Megakaryocyte differentiation was assessed by flow cytometry using fluorescently-labeled antibodies against megakaryocyte- and platelet-specific markers and factor V directly conjugated to a fluorophore over 12 days. Differentiation was confirmed by a decrease in a stem cell marker (CD34) and an increase in a mature megakaryocyte marker (CD42) and coincident with factor V endocytosis. Live cell imaging verified differentiation and permitted the observation of proplatelet formation, the precursor to circulating platelets. Analogous experiments verified the trafficking of factor V into proplatelet extensions.
Factor V is a highly glycosylated protein: potential roles of these glycans may be endocytosis and trafficking by megakaryocytes. We previously demonstrated that factor V endocytosis is mediated by the light chain region of the procofactor. This region of factor V contains three glycans - one high mannose and two complex N-linked glycans. In the second part of this dissertation, a role for the complex N-linked glycans at Asn1675 and Asn2181 of the factor V light chain in factor V endocytosis by megakaryocytes was assessed. Exoglycosidases were used to selectively trim the complex N-linked glycans on human factor V under native conditions. Treatment with neuraminidase removed 100% of the sialic acid residues on the factor V light chain as demonstrated by gel electrophoresis and mass spectrometry. Treatment with β-1,4-galactosidase removed 69% of the galactose residues at Asn1675 and 100% at Asn2181. Glycosidase-treated factor Va behaves similarly to untreated factor Va in thrombin generation assays suggesting that cofactor activity is unaltered by glycan trimming. In addition, glycan removal had no effect on factor V endocytosis by megakaryocyte-like cells. These observations suggest that complex N-linked glycans on the factor V light chain are not important for factor Va cofactor activity or factor V endocytosis by megakaryocyte-like cells, which strongly suggests that they have a role in trafficking.
17
Fock, Ee-Ling Clinical School St George Hospital Faculty of Medicine UNSW. "Molecular regulation and enhancement of megakaryopoiesis and thrombopoiesis by the p45 subunit of NF-E2." Publisher:University of New South Wales. Clinical School - St George Hospital, 2008. http://handle.unsw.edu.au/1959.4/42885.
Full textAbstract:
Megakaryocytes (MKs) are a rare population of haematopoietic cells, which produce platelets. Platelet production is a complex process that is tightly regulated at the transcriptional level by lineage specific transcription factors such as p45 NF-E2. Understanding how transcriptional regulators operate is imperative to advance our knowledge of disease pathophysiology and to propose novel treatment options. Therefore, the aims of this study were to: i) study the effects of p45 NF-E2 overexpression on various stages of megakaryopoiesis; (ii) elucidate the nuclear transport mechanisms of p45 NF-E2; and iii) determine the impact of a p45 NF-E2 modification called SUMOylation on thrombopoiesis. Exogenous p45 NF-E2 was overexpressed in haematopoietic cells in culture and various aspects of megakaryopoiesis were examined. Overexpression of p45 NF-E2 enhanced multiple stages of MK differentiation such as colony forming unit (CFU)-MK formation and terminal MK maturation. Most importantly, p45 NF-E2 overexpression resulted in significant increases in proplatelet and functional platelet production in vitro. This latter result was confirmed in vivo using lethally irradiated mice transplanted with cells that overexpressed p45 NF-E2. Unexpectedly, the enhancement of MK differentiation was at the expense of myeloid development and, for the first time, identified p45 NF-E2 as a negative regulator of myeloid differentiation. Secondly, we determined the nuclear localisation signal of p45-NF-E2 and the pathway responsible for nuclear import. We also investigated the importance of p45 NF-E2 nuclear import in thrombopoiesis. Finally, we showed that p45 NF-E2 is modified mainly by SUMO-2/3 in bone marrow cells and this process is involved in the transcriptional activation of MK-specific genes and platelet release. Taken together, these results suggest that enforced expression of p45 NF-E2 selectively enhances many aspects of MK differentiation including early and terminal MK maturation, proplatelet formation and platelet release. Equally important, this thesis also indicates that white blood cell differentiation may be inhibited by p45 overexpression, while molecular processes such as the nuclear import and SUMOylation of p45 NF-E2 are vital for thrombopoiesis. These observations will facilitate subsequent studies into the feasibility of manipulating p45 NF-E2 protein levels for the treatment of conditions such as thrombocytopaenia and other platelet disorders.
18
Emambokus, Nikla R. "Applications of the Cre-LoxP technology to the study of megakaryocytes." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325900.
Full text
19
Schachtner, Hannah. "Investigation of the functional and structural role of podosomes in megakaryocytes." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4032/.
Full textAbstract:
Megakaryocytes (Mks) give rise to platelets via extension of proplatelet arms, which are released through the vascular sinusoids into the bloodstream. Mks and their precursors undergo varying interactions with the extracellular environment in the bone marrow during their maturation and positioning in the vascular niche. The dynamic remodeling of the actin cytoskeleton and the formation of cell -matrix contacts such as podosomes are fundamental for this process. However, the role and function of podosome structures in Mks are poorly understood. Podsomes are well characterized in different cell-types of the myeloid lineage such as macrophages and dendritic cells. Their formation is associated with a dynamic F-actin turnover, fascilitated cell migration and the degradation of extracellular matrix (ECM). The function of podosome organelles is multifaceted and is described in association with cell adhesion, motility, ECM lysis, invasion and mechanosensors. A fundamental analysis of podosomes was necessary to define a potential function for these structures in Mks. I determined an abundant formation of classical podosomes with an F-actin core and a Vinculin ring in primary murine Mks, which were adherent on different physiological relevant ECM substrates such as fibrinogen, collagen I and a native basement membrane. Lifetime analysis was performed and was demonstrated to be dependent on the substrate as well as on Myosin-II activity. Another key feature of podosomes, the degradation of ECM proteins, could be detected and was mediated in an MMP associated manner. Furthermore, I verified that podosomes are necessary to penetrate a native basement membrane, which is amongst others part of blood vessels. II In this thesis I therefore demonstrate multifaceted properties of Mk podosomes and direct a potential function of these structures in the process of Mk maturation and possibly in platelet formation
20
Nürnberg, Sylvia. "Transcription regulation of the megakaryocyte by MEIS1." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610001.
Full text
21
Wang, Jinju. "Differentiation of Megakaryocytes/Platelets and Neurons from Human Endometrial Stromal Progenitor Cells." Wright State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=wright1314976408.
Full text
22
Clarke, Murray Charles Henry. "Cell death in platelets and megakaryocytes : implications for the maintenance of thrombostasis." Thesis, University of Edinburgh, 2002. http://hdl.handle.net/1842/23305.
Full textAbstract:
We now report that thrombostasis is potentially maintained by the relative level of two opposing and different cell death programs within megakaryocytes and platelets. Firstly, platelet formation from megakaryocytes occurs by a unique, compartmentalised form of caspase-dependent apoptosis that results in the formation of multiple functional anucleate progeny. However, whilst the megakaryocyte cell body contains active caspases and displays nuclear condensation and fragmentation typical of apoptosis, the forming platelets retain functional mitochondria, do not contain active caspases, and are not phagocytosed, emphasising an atypical compartmentalised mitochondrial-independent apoptotic program. Platelet formation from megakaryocytes could be significantly augmented by ligation of the Fas death receptor, and reduced by treatment with caspase inhibitors. Secondly, we report a constitutive but caspase-independent program for the specific phagocytic clearance of intact effete platelets. Platelets aged in vitro exhibited increased expression of proapoptotic Bak and Bax, underwent diminution of function, and displayed cytoplasmic condensation and plasma membrane changes that lead to recognition by phagocyte scavenger receptors. In addition, evidence of platelets with an identical morphology was found in patients having sustained a non-Q-wave myocardial infarction. However, although platelets contained the effector caspase-3 they lacked caspase-9, a key component of the apoptosis initiator complex the apoptosome. Intriguingly, megakaryocytes contained both caspase-3 and -9, suggesting sequestration of the enzyme by the progenitors during platelet formation, again underscoring the compartmentalised death of megakaryocytes and thus explaining the inability of platelets to activate caspases during constitutive death, and hence their commitment to undergo a caspase-independent death.
23
Ly, Nam Matthias [Verfasser]. "Influence of megakaryocytes and megakaryocytopoiesis on plasma cell survival / Nam Matthias Ly." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2018. http://d-nb.info/1153769042/34.
Full text
24
Eisbacher, Michael School of Medical Science UNSW. "The regulation of megakaryocyte-specific genes by Fli-1 and GATA-1." Awarded by:University of New South Wales. School of Medical Science, 2003. http://handle.unsw.edu.au/1959.4/19171.
Full textAbstract:
The successive activation of tissue-specific genes during cellular differentiation is orchestrated by the formation of transcriptional complexes consisting of cellspecific and ubiquitous transcription factors. Understanding the molecular events associated with normal megakaryocyte (Mk) differentiation is an issue of central importance to haematology. The aims of this study were therefore to: (i) define the transcription factors responsible for regulating the expression of Mkspecific genes such as Glycoprotein IX, (ii) identify the protein partners of such important Mk-regulatory transcription factors and (iii) examine the mechanisms utilised by these factors to regulate gene expression. First, the regulatory elements in the GPIX promoter required for basal and inducible expression were examined in megakaryoblastic Dami cells stimulated to undergo differentiation. The resulting data suggested that an Ets site in the GPIX promoter binding the Ets-family member Fli-1 was crucial in regulating both constitutive and inducible GPIX expression. Second, a two-hybrid screen of a K-562 cDNA library was used to identify transcription factors that interacted with Fli-1 and were potential regulators of Mk development. Results of this screen identified a novel protein-protein interaction with GATA-1, a previously well-characterised zinc finger transcription factor also implicated in erythroid and Mk development. Mapping of the domains required for the interaction show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. The biological significance of the Fli-1/GATA-1 interaction was demonstrated in transient transfection assays, which resulted in synergistic activation of Mkspecific promoters. Analysis of Fli-1 and GATA-1 expression in a series of erythroleukaemic and megakaryoblastic cell lines demonstrated that the Fli- 1/GATA-1 combination correlates with a Mk-phenotype. Moreover, expression of Fli-1 in K-562 cells (a line rich in GATA-1 but normally lacking Fli-1) induces endogenous GPIX expression. Quantitative mobility shift assays reveal that Fli- 1 and GATA-1 exhibit cooperative DNA-binding in which the binding of GATA-1 to DNA is increased approximately 26 fold in the presence of Fli-1. This data provides a mechanism for the observed transcriptional synergy. In conclusion, this work suggests that Fli-1 and GATA-1 work together through protein-protein interaction and cooperative DNA-binding to activate the expression of genes associated with the terminal differentiation of Mks.
25
Chen, Jianliang, and 陈健良. "The inhibitory effects of human cytomegalovirus on megakaryopoiesis : megekaryocytic cells and bone marrow derived mesenchymal stormal cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/193520.
Full textAbstract:
Thrombocytopenia is one of the most common hematologic presentations of active human cytomegalovirus (HCMV) infection, especially in recipients of allogeneic hematopoietic stem cell transplantations and newborns of congenital HCMV infection. However, mechanisms of HCMV-induced thrombocytopenia have not been well understood. The precursor of circulating platelets – megakaryocyte, is derived from hematopoietic stem/progenitor cell in bone marrow. We postulate that inhibition to megakaryocytic development is the major pathogenesis of HCMV-induced thrombocytopenia. Megakaryocytic cells as well as supportive microenvironment in bone marrow are major targets of HCMV infection. Presented study mainly focused on the impacts of HCMV to megakaryocytic cells and multipotent mesenchymal stromal cells (MSCs) - the precursor of bone marrow stromal cells.
Based on a megakaryocytic cell model challenged by HCMV in vitro, inhibited megakaryocytic endomitosis, proliferation, and cellular expression were respectively demonstrated as decreased polyploidy population, decreased colony formation, and reduced c-Mpl (thrombopoietin receptor) expressing cells. Evoked apoptosis of megakaryocytic cells was also evidenced with increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation after HCMV infection. Involvement of mitochondrial-mediated intrinsic apoptosis was further shown as losing JC-1 fluorescent signal in infected megakaryocytic cells. These results suggest that inhibition induced by HCMV is exerted through multiple processes directly affecting the megakaryopoietic development.
Functional failure of bone marrow microenvironment was demonstrated in bone marrow derived MSCs infected by HCMV in vitro. Suppressed cytokine production, impaired cellular migration, and hindered differentiation of HCMV-infected MSCs were respectively demonstrated by lowered level of stromal cell-derived factor 1 in culture medium, decreased number of cells passed through a porous membrane in a transwell culture, and reduced differentiated cells in either adipogenic or osteogenic induction cultures. Alongside with these changes, HCMV-induced programmed cell death further contributed to the supportive failure. Autophagic cell death in infected MSCs was demonstrated as massive accumulation of vacuoles with double membrane structure and LC-3b II molecules followed by viability loss. De novo apoptosis was also observed as another process of programmed cell death, shown as increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation of infected MSCs. Increased programmed cell death appeared to be associated with extensive HCMV replication in MSCs, which was featured with typical cytopathic morphology, expression of viral tegument protein pp65, and massive accumulation of various viral particles including mature virions. Sustained activation of extracellular signal-regulated kinases likely represented a signal transduction network connecting viral expression or replication with programmed cell death. In a “MSCs-dependent” megakaryopoiesis model, HCMV-infected MSCs failed to support survival and maintenance of megakaryocytic cells. Taken together, these results suggest that active HCMV expression or replication inhibits multiple cellular functions and induces multiple processes of programmed cell death of MSCs. Such inhibition compromises supportive functions of bone marrow microenvironment, and subsequently reduces platelet production in an indirect manner.
In summary, HCMV suppresses cellular function and induced apoptosis on both megakaryocytic cells and their supportive cells, MSCs. Therefore, the inhibitory effects of HCMV on megakaryopoiesis are operated via both direct and indirect mechanisms.published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy
26
Ye, Jieyu, and 叶洁瑜. "The role of platelet-derived molecules: PDGF and serotonin in the regulation of megakaryopoiesis." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47244446.
Full textAbstract:
Investigations on platelet-derived growth factor (PDGF) and serotonin (5-HT), molecules stored in platelet granules, imply their potential effects in regulating megakaryopoiesis, which also intimates the existence of an autocrine and/or paracrine loop constructed by megakaryocytes/platelets and their granular constituents. In addition, numerous reports indicate that melatonin, a derivative from serotonin effectively enhances platelet counts in patients with thrombocytopenia. However, their exact roles on human megakaryocytes and the underlying mechanisms remain unknown.
Present studies showed that PDGF, like thrombopoietin (TPO), significantly promoted platelet recovery and the formation of bone marrow colony-forming unit-megakaryocyte (CFU-MK) in an irradiated-mouse model. An increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis were found in the bone marrow aspirate. In the M-07e apoptotic model, PDGF had a similar anti-apoptotic effect as TPO on megakaryocytes. Our findings demonstrated that PDGF activated the PI3-k/Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression. Our findings suggested that the PDGF-initiated radioprotective effect is likely to be mediated via PDGF receptors (PDGFRs) with subsequent activation of the PI3-k/Akt pathway. We also provide a possible explanation that blockade of PDGFR may reduce thrombopoiesis and play a role in imatinib mesylate-induced thrombocytopenia.
We explored how serotonin regulated megakaryopoiesis and proplatelet formation. Our results indicated that serotonin (5-HT) significantly promoted CFU-MK formation and reduced apoptosis on megakaryocytes through phosphorylation of Akt. These effects were attenuated by addition of ketanserin, a 5-HT2 receptor inhibitor. In addition, serotonin was able to stimulate the F-actin reorganization in megakaryocytes through activating the p-Erk1/2 expression.
Bone marrow mesenchymal stromal cells (MSCs) are important in regulating megakaryopoiesis through stimulating the release of thrombopoietic growth factor, such as TPO. Our studies suggested that when activated by serotonin, bone marrow MSCs were induced to release significant amount of TPO. Furthermore, thousands of membrane-derived microparticles (MPs) arose from MSCs and the TPO RNA/proteins contained within MPs were also considerably increased under serotonin treatment. In summary, our findings demonstrated an important role serotonin played on megakaryopoiesis. This effect was likely mediated via 5HT2 receptors with subsequent activation of Akt and Erk 1/2 phosphorylation, which led to survival of megakaryocytes and proplatelet formation. Serotonin also stimulated TPO released from MSCs in both dissociative and MP-encapsulated form, which indirectly promoted megakaryopoiesis.
The effects of melatonin on megakaryopoiesis were also determined in our studies. Our findings showed that melatonin enhanced proliferation and reduced doxorubicin-induced toxicity on MKs. We further demonstrated the mechanism for melatonin-mediated protection on MKs maybe via repair of G2/M phase cell cycle arrest and inhibition of cell apoptosis on MK cells.
The effects of melatonin on megakaryopoiesis were also determined in our studies. Our findings showed that melatonin enhanced proliferation and reduced doxorubicin-induced toxicity on MKs. We further demonstrated the mechanism for melatonin-mediated protection on MKs maybe via repair of G2/M phase cell cycle arrest and inhibition of cell apoptosis on MK cells.published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy
27
Yau, Yin-chun Mabel. "Studies on melatonin receptors in guinea pig platelets and melatonin actions on human leukemic megakaryoblast MEG-01 cells." Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk/hkuto/record.jsp?B23425039.
Full text
28
Hobbs, Catherine M. "The functional expression of N-methyl-D-aspartate glutamate-type receptors by megakaryocytes and platelets." Thesis, University of Bath, 2010. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.527791.
Full textAbstract:
This study investigated the role of NMDARs in the differentiation of MEG-01 cells and in the activation of human platelets. This investigation demonstrated that the NR1, NR2D and NR3 subunit proteins are expressed in human platelets, with the NR1 subunit also expressed in MEG-01 cells. The NR2A subunit protein was not detectable in either MEG-01 cells or human platelets. PMA-induced differentiation of MEG-01 cells did not appear to stimulate changes in expression of any of the subunit proteins tested. Using assays to measure the changes in [Ca2+]i and ATP secretion, it was determined that donors could be separated into those who responded to the agonists applied and those who did not; responses also decreased over time in both assays. Human platelets from responding donors demonstrate an increase in [Ca2+]i in response to extracellular glutamate, and that increases in ATP secretion are detected at a 10-fold lower concentration. The same is also true with extracellular glycine. Increases in [Ca2+]i were elicited on the addition of extracellular NMDA; extracellular D-serine had no effect. NMDAR inhibitors, MK-801 and D-AP5, inhibited ATP secretion evoked by either glutamate alone or in combination with glycine. D-serine inhibited responses elicited by extracellular glycine. NMDARs play a role in MK differentiation, with the adhesion of MEG-01 cells cultured on a fibrinogen-surface and differentiated with PMA reduced by both inhibitors. PMA-treated MEG-01 cells increased both in size and irregularity, with the addition of NMDAR-specific inhibitors having no effect. S-nitrosylation also inhibits activation of NMDAR, and a new molecule has been developed which can detect S-nitrosylated proteins through a single step process in live cells. Overall, this study has shown that both human platelets and MEG-01 cells express NMDAR subunits, which have been demonstrated to form functional receptors in human platelets.
29
Facchinetti, L. "PLATELET-ASSOCIATED TISSUE FACTOR EXPRESSION: INSIGHTS INTO THE MEGAKARYOCYTE-PLATELET AXIS." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/229418.
Full textAbstract:
Tissue factor is the main activator of the blood coagulation cascade and for this reason levels of TF are not easily detectable in cells in contact with blood under physiological conditions. By contrast, in pathological conditions endothelial cells and monocytes can be induced to express TF. Induced-TF is present in atherosclerotic plaques (vessel wall-derived TF) and several studies in the past have documented its role in the plaque thrombogenicity. At the end of the last century it was reported that, under physiological conditions, TF positive microparticles (MPs) circulate in the blood (blood-borne TF) and their number further increases in pathological conditions. These microparticles, which arise mainly from activated endothelial cells and monocytes, could fuse with other cells conferring them the ability to activate coagulation.
Giesen et al. in 2000 showed the presence of TF in human platelets through this mechanism. Since then several papers have documented the presence of TF in human platelets by using different approaches. Despite this in-depth characterization, the platelet-associated TF is matter of controversy by some authors that, failing to identify TF in platelets, argue that the published data are artifacts, thus generating an ongoing debate.
To date two main “cellular entities” may be responsible for the presence of TF in platelets: TF-positive MPs derived from different activated cell types, as proposed by Nemerson’s group, and megakaryocytes that transferring TF mRNA to platelets make them autonomous in the synthesis of the protein. In this regard, ten years ago our group provided the evidence that human megakaryocytes contain TF mRNA. The platelet transcriptome derives from megakaryocytes through finely tuned mechanisms. The direct evidence that megakaryocytes transfer TF mRNA to platelets, however, is still missing; similarly, it has never been investigated whether megakaryocytes express TF protein that can be transferred to platelets; finally, the contribution of platelet TF to clot formation has never been assessed. To test these hypothesis we took advantage of a well-characterized human megakaryoblastic cell line, Meg-01, able to differentiate into megakaryocytes and to release platelets in vitro. This approach allowed us to analyze TF mRNA and protein expression during the differentiation from megakaryoblasts to megakaryocytes and in the released platelets in the complete absence of any other “contaminating” cell that might be a source of microparticles. The expression of TF protein by Meg-01 and Meg-derived platelet (Meg-platelets) was confirmed in human megakaryocytes differentiated from CD34+ cells. Finally, TF mRNA was silenced in Meg-01 megakaryoblasts showing that megakaryocytes, Meg-platelets and MPs were almost devoid of TF.
Using a combination of cell biology, flow cytometry, confocal microscopy and biochemical analyses here we show evidence for the first time that functionally active TF is expressed in human megakaryocytes, and that they transfer this protein to platelets and to microparticles (MPs). TF downregulation in megakaryoblasts by lentiviral shRNA particles almost completely abolished its expression in megakaryocytes, platelets and MPs. Furthermore, TF silencing leads to a decrease of the thrombin generation by megakaryocytes which is reflected on a decrease in thrombin generation by platelets, suggesting that the platelet-associated TF may contribute to the haemostatic capacity of whole blood.
Taken together all these data shoe the evidence that human platelets and MPs carry a megakaryocyte-derived TF which is functionally active and able to trigger thrombin generation.
Finally, this in vitro model may be used to investigate the modification in platelet transcriptome and functionality observed in several diseases, such as cardiovascular disease, diabetes and cancer.
30
Okyere, Benjamin. "Evaluation of the actin architecture in dysplastic megakaryocytes expressing the NUP98-HOXD13 leukemic fusion gene." Thesis, Virginia Tech, 2013. http://hdl.handle.net/10919/23738.
Full textAbstract:
Some myelodysplastic syndrome (MDS) patients present with macrothrombocytopenia due to impaired megakaryocyte (MK) differentiation. Transgenic mice that express the NUP98-HOXD13 (NHD13) fusion gene is a model for MDS and recapitulates the key features of MDS. The study investigated the hypothesis that expression of NHD13 disrupts actin architecture during MK differentiation leading to macrothrombocytopenia. To test the hypothesis, sternums were stained with hematoxylin and eosin, and evaluated by light microscopy to analyze MK morphology in vivo. NHD13 bone marrow (BM) contained many dysplastic MK. BM from wild type (WT) and NHD13 mice were also flushed, cultured in media supplemented with thrombopoietin only or with estrogen to induce proplatelet formation, and MK harvested after 5 days. Harvested MK and BM cores were processed and analyzed by transmission electron microscopy (TEM) to detail the ultrastructural features. TEM of MK revealed that NHD13 leads to formation of an irregular demarcation membrane system and fewer proplatelets. Cultured WT and NHD13 MK were also cytospun onto glass slides, labeled with fluorescent-tagged F-actin, α/β-tubulin and myosin IIa, and their cytoskeleton compared. Interestingly WT MK had actin either distributed evenly or predominantly in the periphery of the cytoplasm, NHD13 MK displayed only the former phenotype. Additionally, proplatelets lacked actin cytoplasmic extensions. The results from the present thesis demonstrate actin expression and architecture are impaired in dysplastic MK expressing the NHD13 leukemic fusion gene and leads to macrothromcytopenia. Understanding the molecular mechanisms of abnormal MK differentiation in MDS is important as many MDS patients die of hemorrhagic complications.Master of Science
31
Rojnuckarin, Ponlapat. "Mitogen-activated protein kinase pathways in megakaryocyte development /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/9200.
Full text
32
Becker, Isabelle Carlotta [Verfasser], and Bernhard [Gutachter] Nieswandt. "The role of megakaryocytes and platelets in vascular and osteogenic development / Isabelle Carlotta Becker ; Gutachter: Bernhard Nieswandt." Würzburg : Universität Würzburg, 2021. http://d-nb.info/1240614713/34.
Full text
33
Zetterberg, Eva. "Angiogenesis in myeloproliferative disorders /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-383-3/.
Full text
34
游燕珍 and Yin-chun Mabel Yau. "Studies on melatonin receptors in guinea pig platelets and melatonin actions on human leukemic megakaryoblast MEG-01 cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B31242613.
Full text
35
Pathansali, Rohan. "The role of megakaryocytes and platelets in vascular risk factors and vascular disease and the effects of treatment." Thesis, King's College London (University of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.412571.
Full text
36
Mansour, Rana. "Rôle du Ptdlns5P et de PIKfyve dans le contrôle de l'intégrité des granules plaquettaires." Thesis, Toulouse 3, 2016. http://www.theses.fr/2016TOU30089/document.
Full textAbstract:
Les plaquettes jouent un rôle primordial dans le processus d'hémostase. Elles sont générées à partir des mégacaryocytes (MK) présents dans la moelle osseuse. En plus des compartiments vésiculaires classiques de la voie d'endocytose et de dégradation vers les lysosomes, les plaquettes possèdent deux compartiments sécrétoires additionnels, les granules alpha et denses. Ces granules sont générés au cours de la maturation des MK à partir des corps multivésiculaires (MVB) et contiennent des molécules essentielles aux fonctions plaquettaires. Un défaut dans la production ou le remplissage de ces granules est à l'origine de symdromes hémorragiques. Malgré des études montrant l'implication de certaines protéines du trafic vésiculaire, les mécanismes moléculaires qui contrôlent la biogenèse et la maintenance des granules plaquettaires dans les MK ainsi que les mécanismes de tri des cargos qu'ils contiennent, ne sont pas complètement élucidés. Au cours de ces dernières années les phosphoinositides (PI) sont apparus comme des acteurs majeurs du trafic vésiculaire en régulant de la localisation de certaines protéines. Cependant, peu de choses sont connues à ce jour quant au rôle de ces lipides dans la biogenèse et le trafic des granules plaquettaires dans les MK. Au cours de ma thèse, j'ai étudié le rôle d'un des membres de la famille des PI, le phosphatidylinositol 5-phosphate (PtdIns5P), ainsi que deux enzymes responsables de sa synthèse : la 3-phosphatase MTM1 (mutée dans la myopathie centronucléaire, CNM) et la lipide kinase PIKfyve, dans le contrôle de la dynamique des granules. Mes résultats montrent que MTM1 est présente dans les MK et les plaquettes et est localisée en partie sur les granules denses. Cependant, cette phosphatase n'est pas essentielle pour la production et l'activation plaquettaire. En effet, les souris MTM1 KO ne présentent pas de défaut du nombre plaquettaire, ni d'agrégation et de sécrétion suite à une stimulation par la thrombine ou le collagène. Nous montrons la présence d'autres membres de la famille des myotubularines dans les plaquettes et les MK différenciés, ce qui pourrait expliquer une redondance de fonction. De façon intéressante, nous montrons que la détection de MTM1 à partir de faible quantité de sang (<100 ?l) pourrait déboucher sur la mise au point d'un test diagnostic rapide pour la détection de la CNM. Mes travaux ont été focalisés par la suite sur PIKfyve. En utilisant la lignée leucémique mégacaryoblastique MEG-01 différenciée, je montre pour la première fois que le PtdIns5P est localisé dans les compartiments endosomes tardifs ainsi que dans les granules alpha et denses. Dans ces cellules, PIKfyve contrôle plus de 50% du PtdIns5P. De façon remarquable, l'inhibition pharmacologique de PIKfyve ou son invalidation par siRNA entraine une perte d'identité des granules avec la formation de granules élargis qui présentent à la fois des marqueurs de granules denses et alpha et bloque totalement leur mobilité. Ces données ont été confirmées dans des MK primaires de souris. L'addition de PtdIns5P exogène sur les MEG-01 restaure le phénotype normal des granules démontrant que PIKfyve, par l'intermédiaire du PtdIns5P, contrôle l'intégrité des granules qui est donc un phénomène actif et les mécanismes de fusion/fission des vésicules affectant le tri des cargos. De plus, l'inhibition de PIKfyve dans les plaquettes isolées affecte leur agrégation et leur sécrétion, montrant que PIKfyve et le PtdIns5P peuvent agir d'une part lors de la biogénèse des plaquettes dans les MK et d'autre part sur le fonctionnement des plaquettes. Dans leur ensemble, mes travaux placent PIKfyve et son produit lipidique, le PtdIns5P, comme des acteurs majeurs de la maintenance et l'identité des granules plaquettairesPlatelets play a major role in homeostasis processes. They are generated from megakaryocytes (MKs) in the bone marrow. In addition to the classic vesicular compartments of the endocytic and degradation pathway toward lysosomes, platelets have two additional specialized secretory compartments, the dense and alpha granules. These granules are made during MK maturation from multivesicular bodies (MVB) and contain molecules that are essential to platelet functions. Defect in the production of these granules or absence of their cargos is the cause of hemorrhagic syndromes. Despite many studies showing the implication of vesicle trafficking proteins, the molecular mechanisms controlling the biogenesis and maintenance of the granules and cargo sorting are not completely understood. In recent years, phosphoinositides (PIs) have emerged as key actors in vesicular trafficking playing a role of important spatial regulators of many proteins. However, little is known about the role of these lipids in the biogenesis and the trafficking of platelet granules in the MK.During my thesis, I have studied the role of one the member of the PI family, the phosphatidylinositol 5-phosphate (PtdIns5P), and of two enzymes responsible of its synthesis : the 3-phosphatase MTM1(mutated in the Centronuclear myopathy, CNM) and the lipid kinase PIKfyve, in the control of granules dynamic. My results show that MTM1 is present in MK and platelet and that platelet MTM1 localizes in part on dense granules. However, the phosphatase is not mandatory for platelet production and activation. Indeed, the knock-out of MTM1 in mice has no effect on platelet count, aggregation and secretion following thrombin or collagen stimulation. We show the presence of other members of the myotubularins family in platelet and differentiated MK, which can explain a redundancy in functions. Interestingly, we show that MTM1 detection from small amount of blood (<100 ?l) could lead to the development of a rapid diagnostic test for the detection of the CNM. My work was next focalized on PIKfyve. Using the differentiated leukemic megakaryoblastic cell line MEG-01 as a cell model, I showed for the first time that PtdIns5P is localized on late endosome and on alpha and dense granules. In these cells, PIKfyve controls more than 50% of cellular PtdIns5P. Remarkably, pharmacological inhibition of PIKfyve or its invalidation by siRNA leads to a loss of granules identity with the formation of enlarged granules containing both alpha and dense granules markers, and totally blocks their mobility. These data were also confirmed on primary mice MK. Addition of exogenous PtdIns5P on MEG-01 cells restores the normal phenotype of granules showing that PIKfyve, via PtdIns5P, controls granules integrity, an active phenomenon, and the fusion/fission mechanisms that affect cargos sorting. Furthermore, PIKfyve inhibition in isolated platelet affects their aggregation and secretion, showing that PIKfyve and the PtdIns5P may act on the biogenesis of platelets in MK and also on the function of mature platelets. In conclusion, my Ph.D. work shows that PIKfyve and its product PtdIns5P are major actors in platelet granules maintenance and integrity
37
Psaila, Bethan. "Interactions between megakaryocytes and tumour cells in the bone marrow vascular stem cell niche promote tumour growth and metastasis." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/5945.
Full textAbstract:
Specialized bone marrow microenvironments (vascular and osteoblastic 'niches') regulate normal haematopoietic stem/progenitor cells. Recently, the vascular niche has also been implicated as an area for preferential engraftment of malignant cells. The cellular and molecular factors that regulate the vascular niche and, in particular, the role of megakaryocytes are poorly understood. The aim of my work was to investigate the role of megakaryocytes in homing and engraftment of malignant cells to the bone marrow vascular niche using mouse models. C57Bl/6 wild-type and megakaryocyte-deficient, thrombopoietin (TPO)-/- mice were injected with B16 melanoma or EL4 lymphoma cell lines and the megakaryocyte-vascular niche investigated by immunohistochemistry, confocal microscopy, in vitro culture, co-cultures and gene expression by RT-PCR. In wild-type mice injected with B16 melanoma, platelet size and megakaryocyte numbers significantly increased (P<0.02). B16 tumour cells were found to produce the thrombopoietic factors VEGF, SCF and IL11. Bone marrow sinusoids were almost universally surrounded by one of more megakaryocytes tightly abutting the vascular endothelium, forming the megakaryocyte-vascular niche. Metastatic B16 cells were observed in close association with megakaryocytes in the vascular niche, consistent with this being a port of entry to the bone marrow. In TPO-/- mice, tumour growth and metastasis was markedly retarded and no tumour cells were seen in the bone marrow, suggesting that megakaryocytes play a functional role in metastasis. In TPO-/- bone marrow, vessels were more tortuous and larger in diameter (P=0.01); and expression of PF4, TSP1, VEGF and TGFβ was 70%- 90% lower, suggesting that a major proportion of angiogenic regulatory factors is producted by megakaryocytes in the bone marrow in wild-type mice. Furthermore, in wild-type mice, expression of VEGF and TGFβ significantly increased during tumour growth and metastasis while PF4 expression decreased (P<0.05). Megakaryocyte-conditioned medium (MCM) enhanced the proliferation rate of B16 cells (P<0.001) and also was highly chemotactic for B16 cells (P<0.001), an effect mediated by pertussis toxin-sensitive Gi-protein receptors and reduced in the absence of TSP1. Co-culture with B16 cells increased megakaryocyte expression of VEGF, TGFβ and TSP1 and decreased PF4, consistent with the in vivo observations, while cocultured B16 cells displayed increased expression of VEGF and TGFβ and adhesion integrins. Moreover, pretreating B16 cells with MCM prior to tail vein injection enhanced metastatic engraftment. To investigate the role of megakaryocytes in human malignancy, trephine bone marrow biopsies from patients with metastatic carcinoma were examined. Increased megakaryocyte numbers and abnormal megakaryocyte clustering were observed in the majority of patients, suggesting that megakaryocyte-tumour interactions may also occur in the setting of human metastatic disease. In conclusion, my findings suggest that megakaryocytes contribute to the integrity and function of the bone marrow vascular niche and that cellular/molecular cross talk between megakaryocytes and tumour cells may promote metastasis. Targeting these interactions may be useful as adjunctive therapy in metastatic disease.
38
Lemieux, Justin Michael. "Mechanisms of Hematopoietic-Mesenchymal Cell Activation." Yale University, 2009. http://ymtdl.med.yale.edu/theses/available/etd-03112009-191829/.
Full textAbstract:
As the prevalence of osteoporosis is expected to increase over the next few decades, the development of novel therapeutic strategies to combat this disorder becomes clinically imperative. These efforts draw extensively from an expanding body of knowledge pertaining to the physiologic mechanisms of skeletal homeostasis. To this body of knowledge, we contribute that cells of hematopoietic lineage may play a crucial role in balancing osteoblastic bone formation against osteoclastic resorption. Specifically, our laboratory has previously demonstrated that megakaryocytes can induce osteoblast proliferation in vitro, but do so only when direct cell-to-cell contact is permitted. To further investigate the nature of this interaction, we have effectively neutralized several adhesion molecules known to function in the analogous interaction of megakaryocytes with another cell-type of mesenchymal origin - the fibroblast. Our findings implicate the involvement of fibronectin/RGD-binding integrins including á3â1 (VLA-3) and á5â1 (VLA-5) as well as glycoprotein IIb (CD41), all of which are known to be expressed on megakaryocyte membranes. Furthermore, we demonstrate that IL-3 can enhance megakaryocyte-induced osteoblast activation in vitro, as demonstrated in the megakaryocyte-fibroblast model system. Taken together, these results suggest that although their physiologic and clinical implications are very different, these two models of hematopoietic-mesenchymal cell activation are mechanistically analogous.
39
Sime, Wondossen. "The diverse role of laminin isoforms in neuronal cells, human mast cells and blood platelets /." Stockholm : Karolinska institutet, 2007. http://diss.kib.ki.se/2007/978-91-7357-122-7/.
Full text
40
Johnson, Lacey Nicole St George Clinical School UNSW. "Molecular regulation of Megakaryopoiesis: the role of Fli-1 and IFI16." Awarded by:University of New South Wales. St George Clinical School, 2006. http://handle.unsw.edu.au/1959.4/26819.
Full textAbstract:
Megakaryocytes (Mks) are unique bone marrow cells, which produce platelets. Dysregulated Mk development can lead to abnormal platelet number and the production of functionally defective platelets, causing bleeding, thrombotic events, and leukaemia. Understanding the molecular mechanisms driving megakaryopoiesis may yield insights into the molecular genetics and cellular pathophysiology of a diversity of disorders. The primary aim of this thesis was to gain insight into the molecular events required for normal Mk development. As transcription factors and cytokines play a central role in driving Mk development, both of these processes were investigated. Fli-1 and GATA-1 are key transcription factors regulating Mk-gene expression, alone and co-operatively. To understand the mechanism of transcriptional synergy exerted by Fli-1 and GATA-1, in vitro assays were carried out investigating the interactions between Fli-1, GATA-1 and DNA that mediate synergy. A novel mechanism of synergy was identified, where Fli-1 DNA binding is not required, although an interaction between Fli-1 and GATA-1, and GATA-1 DNA binding is required. Importantly, the results demonstrate that Fli-1 DNA binding is not essential for promoting Mk-gene expression in primary murine bone marrow cells. Thrombopoietin (TPO) is the primary cytokine responsible for Mk and platelet development. Identifying novel TPO gene-targets may provide invaluable information to aid the understanding of the complex and unique processes required for Mk development. Using microarray technology, IFI16 was identified as a TPO-responsive gene that has not previously been studied in the Mk lineage. This work demonstrated that IFI16 is expressed in CD34+ HSC-derived Mks, and that the Jak/STAT pathway is essential for the activation of IFI16 by both TPO and IFN-??. Of biological significance, IFI16 was found to regulate both the proliferation and differentiation of primary Mks, suggesting that IFI16 may control the balance between these two essential processes. In conclusion, the data in this thesis presents a novel mechanism through which Fli-1 and GATA-1 regulate the synergistic activation of Mk genes. The identification and functional characterisation of a novel TPO-inducible gene, IFI16, involved in regulating the proliferation and differentiation of Mks is also described. These findings have implications for several congenital and malignant conditions affecting Mk and platelet development, and possibly a mechanism for IFN-induced thrombocytopaenia.
41
Choudry, Fizzah Aziz. "Novel insights into megakaryopoiesis, thrombopoiesis and acute coronary thrombosis : transcriptome profiling of the haematopoietic stem cell, megakaryocyte and platelet." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/283252.
Full textAbstract:
The aim of this project was to investigate the transcriptome of human haematopoietic stem cells (HSCs), megakaryocytes and platelets to gain insights into steady state and accelerated thrombopoiesis that occurs in states of haemostatic demand and in thrombosis by applying these findings to the pathological setting of acute coronary thrombosis. To investigate transcriptional heterogeneity within the human HSC population, single cell RNA sequencing was performed in human bone marrow HSCs. Transcriptionally distinct subpopulations were identified including two megakaryocyte biased subsets with potentially differing functional relevance. Both populations expressed megakaryocyte specific transcripts, one of which also co-expressed common myeloid and megakaryocyte-erythroid progenitor transcripts while the other did not. This study represents the first interrogation of the human bone marrow megakaryocyte transcriptome. Cells were collected from healthy human bone marrow and analysed by low input and single cell RNA sequencing. To identify novel drivers of megakaryocyte maturation, the human bone marrow megakaryocyte transcriptome was compared to that of megakaryocytes cultured from human CD34+ cells, a process known to generate immature megakaryocytes. Transcriptional signatures associated with increasing megakaryocyte ploidy were then investigated. Increasing megakaryocyte ploidy level was found to be associated with an upregulation of transcripts involved in translation and protein processing as well as expression of a number of transmembrane receptors which might have functional relevance. Finally, the pathological setting of acute coronary thrombosis was used as a model for accelerated thrombopoiesis. Megakaryocyte and platelet transcriptomes were compared between patients with acute myocardial infarction (AMI) as well as severe coronary disease and a control group. The transcriptional signature relating to disease compared to control in megakaryocytes included upregulation of platelet activation related transcripts in megakaryocytes isolated from patients with AMI and severe coronary artery disease.
42
Bennett, Cavan. "Cytokine receptor-like factor 3 (CRLF3) : a novel regulator of platelet biogenesis and potential drug target for thrombocythaemia." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/277068.
Full textAbstract:
Thrombocythaemia is defined as a circulating platelet count above 450x10$^9$/L in humans. The major cause of thrombocythaemia is reactive $(secondary)$ thrombocythaemia which occurs secondary to many conditions such as infection, cancer and inflammation. However, acquired clonal mutations in mainly Janus Kinas 2 $(JAK2)$, CALR and MPL cause essential thrombocythaemia $(ET)$. ET is a rare disease that leads to an increased risk of cardiovascular thrombotic events. Current treatment of ET uses combination of low dose aspirin to decrease platelet function and cytoreductive agents to decrease thrombopoiesis. The most commonly used cytoreductive agents are hydroxyurea, anagrelide and interferon-$alpha$ and all have unwanted side effects. Cytokine receptor-like factor 3 $(CRLF3)$ is a 2.4kb gene that is ubiquitously expressed throughout the haematopoietic system. Very little is known about the function of CRLF3, with only one peer reviewed journal article in the literature which shows that CRLF3 may negatively regulate the cell cycle at the G0/G1 phase. However, nothing is known about the role of CRLF3 in platelet biology. Using a Crlf3 knockout mouse $(Crlf3-/-)$ developed by the Wellcome Trust Sanger Institute we show CRLF3’s role in platelet biogenesis and how it could be used as a novel therapeutic target to treat ET. Crlf3-/- mice have an isolated and sustained 25-40$\%$ decrease in platelet count compared to wildtype $(WT)$ controls. Platelet function is unaffected as demonstrated in a range in a range of in vitro assays. The thrombocytopenia is a consequence of abnormalities in hematopoietic cells, as shown by bone marrow transplantations. Megakaryopoiesis is upregulated in Crlf3-/- mice and proplatelet morphology is unaffected, suggesting the thrombocytopenia is due to increased platelet clearance. Indeed, splenectomised Crlf3-/- mice show normalised platelet counts within 7 days, showing rapid splenic removal of platelets is responsible for the thrombocytopenia. Abnormal large platelet structures that resemble proplatelets shafts $(preplatelets)$ are abnormally present in the circulation of elderly Crlf3/- mice. Immunohistochemistry showed increased and aberrant tubulin expression in Crlf3-/- platelets compared to WT controls, especially in the preplatelet forms. Cold induced depolymerisation of microtubules was decreased in Crlf3-/- platelets, suggestive of increased tubulin stability, however, the ratio of detyrosinated to tyrosinated tubulin was not altered. We then crossbred Crlf3-/- mice with JAK2 V617F ET mice, to determine the effect of Crlf3 ablation of thrombocythaemia. Crossbred mice showed restoration of platelet counts to WT values without grossly affecting platelet function or other blood lineages, providing the rational for CRLF3 as a novel therapeutic target for treatment of ET. Finally, we aimed to resolve the crustal structure of CRLF3 and discover its interactome. To this end, we were able to resolve the crystal structure of a C-terminal portion of the full length protein containing the predicted fibronectin type III domain. To shed light on the interactome of CRLF3, endogenous CRLF3 was tagged with a tandem affinity purification $(TAP)$ tag using CRISPR/Cas9 technology in induced pluripotent stem cells $(iPSCs)$. We have been able to produce megakaryocytes from these TAP-tagged iPSCs by forward programming. However, as yet we have not been able to generate enough MKs to have adequate material to perform immunoprecipitation assays. Therefore, the interactome of CRLF3 in MKs remains unknown. In conclusion, we identified a mechanism by which Crlf3 controls platelet biogenesis. Slowed maturation of Crlf3-/- preplatelets in the peripheral circulation potentially due to increased structural stability leads to rapid removal of these immature forms by the spleen and therefore a decrease in platelet count. The isolated effect on platelet numbers and normalisation of platelet count in ET mice deficient of Crlf3 provides the rational for further study on CRLF3 drug targeting as a novel therapeutic strategy for ET.
43
Arabanian, Laleh Sadat. "Role of NFAT (Nuclear Factor of Activated T Cells) Transcription Factors in Hematopoiesis." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-99739.
Full textAbstract:
Understanding the transcriptional mechanisms that control hematopoiesis and the interaction between hematopoietic stem cells and the bone marrow (BM) microenvironment in vivo is of considerable interest. The calcineurin-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) is known as master regulator of cytokine production in T lymphocytes and therefore central for T cell-dependent immune reactions, but has also been shown to regulate a process of differentiation and tissue adaptation in various cell types. The activation of NFAT is dependent on the calcium level within the cell. In resting cells, calcium levels are low and NFAT is cytoplasmic and inactive. A sustained increase in the internal calcium concentration within an external stimuli leads to activation of the calcium-dependent calcineurin, followed by dephosphorylation and nuclear translocation of NFAT. We have previously shown that NFATc2, a member of the NFAT family, is expressed in CD34+ hematopoietic stem cells (HSC). A mouse model harboring NFATc2 deficiency provides the opportunity for in vivo investigation of the role of NFATc2 in hematopoiesis.
Our recent observations showed that aged mice lacking the transcription factor NFATc2 develop peripheral blood anemia and thrombocytopenia, BM hypoplasia and extramedullary hematopoiesis in spleen and liver. The proliferation and differentiation of NFATc2-deficient hematopoietic stem cells ex vivo, however, was found to be intact. It remained therefore unclear whether the disturbed hematopoiesis in NFATc2-deficient mice was caused by the hematopoietic or the stroma component of the BM hematopoietic niche. In the current study we dissected the relative contribution of hematopoietic and stroma cells to the phenotype of the NFATc2-deficent mice by transplanting immuno-magnetically purified NFATc2-deficient (KO) HSCs to lethally irradiated wild type (WT) mice, and vice versa. After a post-transplantation period of 6-8 months, peripheral blood, BM as well as spleen and liver of the transplanted animals were analyzed and compared to WT and KO mice transplanted with control cells.
Transplantation of NFATc2-deficient HSCs into WT recipients (KO WT) induced similar hematological abnormalities as those occurring in non-transplanted KO mice or in KO mice transplanted with KO cells (KO KO). Compared to WT mice transplanted with WT cells (WT WT), KO WT mice showed evidence of anemia, thrombocytopenia and a significantly reduced number of hematopoietic cells in their BM. Likewise, KO WT mice developed clear signs of extramedullary hematopoiesis in spleen and liver, which was not the case in WT WT control animals. In addition to the hematopoietic abnormalities, transplantation of NFATc2-deficient HSC also induced osteogenic abnormalities such as BM sclerosis and fibrosis in WT mice. This phenomenon was rather subtle and of incomplete penetrance, but never seen in mice transplanted with WT cells. These data demonstrate for the first time, that the NFATc2 transcription factor directly regulates the intrinsic function of hematopoietic stem cells in vivo. However, the transcriptional targets for NFAT in these cells are yet unknown. In addition to hematopoietic stem cells, NFATc2 has been shown to be expressed in a lineage-specific manner during myeloid differentiation and, notably, is maintained during megakaryopoiesis while it is suppressed during the differentiation of neutrophils. Bone marrow megakaryocytes are the precursors of peripheral blood platelets and therefore constitute an integral part of primary hemostasis, thrombosis and wound healing. The biological role of NFAT in megakaryocytes is unknown. We have recently shown that NFATc2 is not necessary for megakaryocytic differentiation. On the other hand, recent evidence suggests that NFATc2 is required for the transcription of specific megakaryocytic genes. In this study, we showed that activation of the calcineurin/NFAT pathway in either primary megakaryocytes or CMK megakaryocytic cells forces the cells to go into apoptosis. Cell death in megakaryocytes is induced by treating the cells with the calcium ionophore ionomycin and suppressed by either the pan-caspase inhibitor zVAD or the calcineurin inhibitor cyclosporin A (CsA). Ionomycin stimulation of megakaryocytes leads to the expression of Fas Ligand (FASLG), a pro-apoptotic member of the tumor necrosis factor superfamily. Expression of FASLG was detectable as early as four hours after stimulation on the membrane of ionomycin-treated megakaryocytes, was augmented in cells stably overexpressing NFATc2, and was suppressed in cells either pretreated with CsA or expressing the specific peptide inhibitor of NFAT, VIVIT.
To investigate the physiological relevance of FASLG expression on megakaryocytes, we performed co-cultures of megakaryocytes with Fas-expressing T-lymphocytes, in which CMK cells were left either unstimulated or pre-stimulated with ionomycin and then added to Jurkat cells. The presence of ionomycin-stimulated CMK cells, but not of unstimulated cells or cells stimulated in the presence of CsA, significantly induced apoptosis in Jurkat cells. Overexpression of NFATc2 in CMK cells enhanced their potency to induce apoptosis in Jurkat cells, while cells expressing VIVIT were less effective. Apoptosis induction of Jurkat cells by stimulated CMK cells was partially blocked by the presence of either a neutralizing antibody against FASLG or an antagonistic antibody to Fas during the co-culture period, indicating involvement of the FASLG/Fas apoptosis pathway. These results represent the first clear evidence for a biological function of the calcineurin/NFAT pathway in megakaryocytes, namely the regulation of Fas/FASLG-dependent apoptosis. Second, they underline that the biological role of megakaryocytes is not restricted to the production of proteins and other cellular structures for platelet assembly, but that this population of cells fulfills an independent regulatory function in the context of the surrounding tissue.
Finally, we have identified by RNA sequencing analysis of NFATc2-expressing and -deficient cells, the entire set of genes which is induced by NFATc2 in stimulated megakaryocytes. Functional pathway analysis suggests an involvement of NFATc2 in pro-inflammatory pathways in these cells. The significance of these findings has to be addressed in further studies.
44
Gerner, Frank [Verfasser], Bernhard [Gutachter] Nieswandt, Katrin [Gutachter] Heinze, Christoph [Gutachter] Kleinschnitz, and Antje [Gutachter] Gohla. "Functional analysis of polarization and podosome formation of murine and human megakaryocytes / Frank Gerner ; Gutachter: Bernhard Nieswandt, Katrin Heinze, Christoph Kleinschnitz, Antje Gohla." Würzburg : Universität Würzburg, 2019. http://d-nb.info/1196971595/34.
Full text
45
Desponts, Caroline. "The role of Src homology 2 domain containing 5' inositol phosphatase 1 (SHIP) in hematopoietic cells." [Tampa, Fla] : University of South Florida, 2006. http://purl.fcla.edu/usf/dc/et/SFE0001591.
Full text
46
Schmithausen, Patrick Alexander Gerhard [Verfasser], Katrin [Gutachter] Heinze, Andreas [Gutachter] Beilhack, and Bernhard [Gutachter] Nieswandt. "Three-dimensional fluorescence image analysis of megakaryocytes and vascular structures in intact bone / Patrick Alexander Gerhard Schmithausen ; Gutachter: Katrin Heinze, Andreas Beilhack, Bernhard Nieswandt." Würzburg : Universität Würzburg, 2019. http://d-nb.info/1182230369/34.
Full text
47
Al, Maghout Tamer [Verfasser]. "P38 Kinase, SGK1 and NF-κB Dependent Up-Regulation of Na+/Ca2+ Exchanger Expression and Activity Following TGFß1 Treatment of Megakaryocytes / Tamer Al Maghout." Tübingen : Universitätsbibliothek Tübingen, 2020. http://d-nb.info/1221596780/34.
Full text
48
Barnes, Calvin Langston Toure. "C-mpl Expression in Osteoclast Progenitors: A Novel Role for Thrombopoietin in Regulating Osteoclast Development." Yale University, 2006. http://ymtdl.med.yale.edu/theses/available/etd-06262006-123750/.
Full textAbstract:
A new paradigm has evolved in which multiple regulatory interactions between the skeletal and hematopoietic systems have been identified. Previous studies have demonstrated that megakaryocytes (MK) play a dual role in skeletal homeostasis by stimulating osteoblast proliferation and simultaneously inhibiting osteoclast (OC) development. Here we identify a novel regulatory pathway in which the main MK growth factor, thrombopoietin (TPO), directly regulates osteoclastogenesis. To study the role of TPO in OC development, spleen or bone marrow (BM) cells (2x10[exponent]6 cells/ml) or BM macrophages (BMM, 1x10[exponent]5 cells/ml) from C57BL/6 mice , as a source of OC precursors, were cultured with M-CSF (30 ng/ml) and RANKL (50 ng/ml) to induce OC formation. TPO (0.1-1000 ng/ml) and/or primary MK (0-0.5%), derived from C57BL/6 fetal livers, were titrated into these cultures and OC were identified as tartrate resistant acid phosphatase positive (TRAP+) giant cells with >3 nuclei. There was a significant, up to 15-fold reduction in OC formed when MK were added to all OC generating cultures, p < 0.001. Moreover, if OC generating cultures did not contain MK or MK progenitors, TPO treatment significantly enhanced OC formation up to six-fold, p < 0.01. This data demonstrates that MK are responsible for the inhibition of OC formation and that in cultures containing MK or MK progenitors such as BM or spleen cells, that TPO acts indirectly to inhibit OC formation by stimulating megakaryopoiesis, whereas in the absence of MK or MK progenitors TPO directly enhances OC formation. This conclusion is further supported by Real-Time PCR data which demonstrates that OC progenitors express c-mpl, the TPO receptor, albeit at low levels when compared to expression of c-mpl on MK. Finally, we have begun to dissect the c-mpl signaling pathway in OC progenitors. We have found that TPO induces tyrosine phosphorylation of several specific cellular proteins in the JAK/STAT pathway. Thus, TPO acts in a somewhat paradoxical manner by inhibiting OC formation through the stimulation of MK, while simultaneously playing a direct role in enhancing osteoclastogenesis.
49
Watanabe, Caroline Mitiká. "Avaliação da infecção de megacariócitos e plaquetas pelo VHC e sua influência na fisiopatologia da hepatite C." Botucatu, 2016. http://hdl.handle.net/11449/138765.
Full textAbstract:
Orientador: Paulo Eduardo de Abreu MachadoResumo: A hepatite C acomete cerca de 130-150 milhões de pessoas em todo o mundo, sendo que grande parcela dos portadores do vírus da hepatite C (VHC) permanecem assintomáticos por longos períodos. O diagnóstico da doença acontece muitas vezes devido a sintomas extra-hepáticos como fadiga crônica, alterações endócrinas, dermatológicas e hematológicas, porém, a patogênese das manifestações extra-hepática é pouco conhecida. Assim, modelos que reproduzam a infecção in vitro pelo VHC se tornam necessários para que se possa compreender e esclarecer a relação entre estas desordens e o VHC. O VHC é um Hepacivírus da família Flaviviridae, sua entrada em células suscetíveis, como os hepatócitos, pode ocorrer por infecção direta, mediada principalmente pelos receptores CD81 e Claudina-1 (CLDN1), desencadeando uma série de processo para internalização e replicação viral, ou por infecção por contato célula-a-célula, mediada por CLDN1 e Ocludina (OCLN), não sendo necessário, por esta via, CD81. Estudos evidenciam a interação entre plaquetas e VHC, no entanto, não demonstram claramente se estas células somente aderem às partículas virais ou se são infectadas pelo vírus. Assim, estudos que contribuam à elucidação deste processo são salutares. Desta forma, o objetivo deste trabalho foi avaliar a infecção de megacariócitos e plaquetas pelo VHC e verificar a influência na fisiopatologia da hepatite C. Amostras de megacariócitos, provenientes de doadores de medula óssea, e amostras de plaquetas perif... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Hepatitis C affects about 130-150 million people worldwide. It´s caused by Hepacivirus and the diagnosis are by extrahepatic symptoms such as chronic fatigue, endocrine, dermatologic and hematologic changes. However, the pathogenesis of extrahepatic manifestations is little known and requires further studies on the relationship of these disorders and the hepatitis C vírus (HCV), therefore infection in vitro model can improve this kind of knowledge. HCV is a family of Flaviviridae, its entry into susceptible cells, such as hepatocytes, can occur by direct infection mediated primarily by CD81 receptors and Claudin-1 (CLDN1), triggering a process serie for internalization and viral replication ocurr. Other road is by cell-to-cell mediated CLDN1 and occludin (OCLN), not being necessary the presence of CD81. Studies describe the interaction between platelets and HCV, however, does not clearly denotes how this process happens, due to this, studies can contribute to the elucidation of this process relevance. The aim of this study is the evaluation of the infection of megakaryocytes and platelets HCV and the influence in pathophysiology of hepatitis C. Megakaryocytes samples, from bone marrow donors, and samples of peripheral platelets, both obtained from healthy donos are infected in vitro with HCV positive plasma. Infected samples were evaluated by flow cytometry and confocal microscopy. The parameters analyzed were the presence or absence of viral and expression of CLDN1 and CD81... (Complete abstract click electronic access below)
Mestre
50
Sundaramoorthi, Hemalatha. "Identification of Hox Genes Controlling Thrombopoiesis in Zebrafish." Thesis, University of North Texas, 2015. https://digital.library.unt.edu/ark:/67531/metadc822768/.
Full textAbstract:
Thrombocytes are functional equivalents of mammalian platelets and also possess megakaryocyte features. It has been shown earlier that hox genes play a role in megakaryocyte development. Our earlier microarray analysis showed five hox genes, hoxa10b, hoxb2a, hoxc5a, hoxc11b and hoxd3a, were upregulated in zebrafish thrombocytes. However, there is no comprehensive study of genome wide scan of all the hox genes playing a role in megakaryopoiesis. I first measured the expression levels of each of these hox genes in young and mature thrombocytes and observed that all the above hox genes except hoxc11b were expressed equally in both populations of thrombocytes. hoxc11b was expressed only in young thrombocytes and not in mature thrombocytes. The goals of my study were to comprehensively knockdown hox genes and identify the specific hox genes involved in the development of thrombocytes in zebrafish. However, the existing vivo-morpholino knockdown technology was not capable of performing such genome-wide knockdowns. Therefore, I developed a novel cost- effective knockdown method by designing an antisense oligonucleotides against the target mRNA and piggybacking with standard control morpholino to silence the gene of interest. Also, to perform knockdowns of the hox genes and test for the number of thrombocytes, the available techniques were both cumbersome or required breeding and production of fish where thrombocytes are GFP labeled. Therefore, I established a flow cytometry based method of counting the number of thrombocytes. I used mepacrine to fluorescently label the blood cells and used the white cell fraction. Standard antisense oligonucleotide designed to the central portion of each of the target hox mRNAs, was piggybacked by a control morpholino and intravenously injected into the adult zebrafish. The thrombocyte count was measured 48 hours post injection. In this study, I found that the knockdown of hoxc11b resulted in increased number of thrombocytes and knockdown of hoxa10b, hoxb2a, hoxc5a, and hoxd3a showed reduction in the thrombocyte counts. I then screened the other 47 hox genes in the zebrafish genome using flow sorting method and found that knockdown of hoxa9a and hoxb1a also resulted in decreased thrombocyte number. Further, I used the dye DiI, which labels only young thrombocytes at specific concentrations and observed that the knockdown of hoxa10b, hoxb2a, hoxc5a, hoxd3a, hoxa9a and hoxb1a, lead to a decrease in young thrombocytes; whereas hoxc11b knockdown lead to increase in number of young thrombocytes. Using bromodeoxyuridine, I also showed that there is increase in release of young thrombocytes into peripheral circulation in hoxc11b knockdown fish which suggests that hoxc11b significantly promotes cell proliferation rather effecting apoptosis. In conclusion, I found six hox genes that are positive regulators and one hox gene is a negative regulator for thrombocyte development.